Project Summary/Abstract Human immunodeficiency virus (HIV) infects 37 million people globally and has killed over 39 million since its emergence in the human population. There are two different types of HIV: type 1 (HIV-1) and type 2 (HIV-2), with the latter exhibiting an attenuated disease phenotype that has been recognized for decades. A high rate of mutation is a hallmark feature of HIV-1 disease progression and drives immune evasion as well as the emergence of drug resistance. While the majority of viral mutagenesis is thought to be driven by the low fidelity of the virally-encoded reverse transcriptase (RT), restrictions factors that act during reverse transcription can contribute to viral mutation as well. For example, the apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3 (APOBEC3; A3) family of proteins has been identified as a driver of G-to-A mutations in HIV- 1 through deamination of cytosines to uracils during reverse transcription. Another host factor, sterile ? motif domain and histidine-aspartic domain-containing protein 1 (SAMHD1), restricts viral replication by depleting cellular dNTP pools, and could drive viral mutagenesis as well. How these factors impact HIV-2 mutagenesis has, to date, been uncharacterized. This proposal seeks to investigate cellular sources of HIV-2 mutagenesis relative to that of HIV-1, and correlate this with differences in virus infectivity. Levels of restriction by A3 proteins will be evaluated by defining the restrictive capacity of the human A3 repertoire against HIV-2, as well differences in the effectiveness of the HIV accessory protein Vif. The contribution of SAMHD1 to HIV-1 and HIV-2 mutagenesis will be determined using high-throughput Illumina sequencing of WT HIV-1 and HIV-2 viruses in monocytic cell lines, as well as with Vpx-deficient HIV-2. Viral mutagens and inhibitors of ribonucleotide reductase will be used to correlate mutagenesis with viral infectivity in order to shed light on how tolerance to mutations impacts viral mutagenesis and infectivity. Taken together, these experiments will serve to determine the relative impact of cellular restriction factors HIV-2 mutagenesis compared to HIV-1 mutagenesis. These comparative studies should provide new insights into the host determinants of HIV mutagenesis.